NK-1 is one of five homeobox genes clustered in the 93E1-5 region of the third chromosome of Drosophila. Previous studies on the expression pattern of NK-1 using in situ hybridization, suggested that NK-1 may be regulated in a tissue-specific manner and may be involved in muscle segment formation and/or neuro-muscular synaptogenesis. In order to understand the function of the NK-1 homeobox gene in neuro-muscular synaptogenesis, we initiated experiments to characterize the cis-acting DNA elements controlling the tissue-specific expression of NK-1 in cultured cells using transient expression assays. From the analysis of 4 kb of the 5' upstream region of NK-1, we found a strong enhancer region which is active in cultured myoblast cells and a silencer region which is active both in muscle cells and in neuronal cells. DNaseI footprinting assays and mutational analysis of this enhancer region defined a novel binding site for unknown factors which are necessary and sufficient to demonstrate enhancer activity in C2C12 myoblast cells. Within the silencer region we found that multiple cis-acting elements are involved in the downregulation of NK-1. These results suggest that the cell-specific expression of the NK-1 homeobox gene in myoblast and neuroblastoma cells may be regulated through both silencer and enhancer elements. Since homeobox genes encode DNA binding proteins that act as transcription factors, one of the ways to understand function of the NK-1 homeodomain transcription factor is to identify target genes regulated by NK-1. For this purpose, we screened for genes that encode proteins which are involved in protein-protein interactions with the NK-1 protein using the yeast two-hybrid system which is a genetic method to detect protein-protein interactions. From the screening of a Drosophila adult match-maker cDNA library, we identified eight positive clones that showed both growth on His-deficient media and beta-galactosidase activity. DNA sequence analysis revealed that these clones could be classified into five groups of novel genes. Using positive clones and various bait constructs containing a different coding region of the NK-1 gene, we found that the middle region of the NK-1 protein (a.a. 150-417) was involved in protein-protein interaction. It is of note that a prd-repeat (alternating His-Gln or His-Pro residues), that is also found in other homeodomain transcription factors and whose function is still unknown, is located within this region. Characterization of these clones is underway.